Mycobiology

The Korean Society of Mycology (한국균학회)
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Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin

  • Jeong, Min-Hye (Department of Plant Medicine, Sunchon National University) ;
  • Kim, Jung A. (Microorganism Resources Division, National Institute of Biological Resources) ;
  • Kang, Seogchan (Department of Plant Pathology & Environmental Microbiology, The Pennsylvania State University) ;
  • Choi, Eu Ddeum (Department of Plant Medicine, Sunchon National University) ;
  • Kim, Youngmin (Department of Plant Medicine, Sunchon National University) ;
  • Lee, Yerim (Department of Plant Medicine, Sunchon National University) ;
  • Jeon, Mi Jin (Microorganism Resources Division, National Institute of Biological Resources) ;
  • Yu, Nan Hee (Department of Agricultural Chemistry, Institute of Environmentally Friendly Agriculture, Chonnam National University) ;
  • Park, Ae Ran (Department of Agricultural Chemistry, Institute of Environmentally Friendly Agriculture, Chonnam National University) ;
  • Kim, Jin-Cheol (Department of Agricultural Chemistry, Institute of Environmentally Friendly Agriculture, Chonnam National University) ;
  • Kim, Soonok (Microorganism Resources Division, National Institute of Biological Resources) ;
  • Park, Sook-Young (Department of Plant Medicine, Sunchon National University)
  • Received : 2021.02.22
  • Accepted : 2021.07.21
  • Published : 2021.10.31
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Abstract

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pretreatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

Keywords

Acknowledgement

This work was supported by the National Institute of Biological Resources, funded by the Ministry of Environment of the Republic of Korea (projects NIBR201921101 and NIBR202021101) and by the Ministry of Science and ICT, Korea, under the Grand Information Technology Research Center support program [IITP-2021-2020-0-01489] supervised by the IITP. The USDA National Institute of Food & Agriculture and Federal Appropriations (Project PEN04655; Accession # 1016291) supported S. Kang

References

  1. Kirk P, Cannon P, Minter D, et al. Dictionary of the fungi. 10th ed. Wallingford (UK): CABI Publishing; 2008.
  2. Fournier J, Flessa F, Persoh D, et al. Three new Xylaria species from southwestern Europe. Mycol Progr. 2011;10(1):33-52. https://doi.org/10.1007/s11557-010-0671-8
  3. Stadler M, Kuhnert E, Persoh D, et al. The xylariaceae as model example for a unified nomenclature following the "one Fungus-One name"(1F1N) concept. Mycology. 2013;4:5-21.
  4. Song F, Wu SH, Zhai YZ, et al. Secondary metabolites from the genus Xylaria and their bioactivities. Chem Biodivers. 2014;11(5):673-694. https://doi.org/10.1002/cbdv.201200286
  5. Edwards RL, Maitland DJ, Pittayakhajonwut P, et al. Metabolites of the higher fungi. Part 33. 1 grammicin, a novel bicyclic C7H6O4 furanopyranol from the fungus Xylaria grammica (mont.) Fr. J Chem Soc, Perkin Trans 1. 2001;11:1296-1299.
  6. Kim TY, Jang JY, Yu NH, et al. Nematicidal activity of grammicin produced by Xylaria grammica KCTC 13121BP against Meloidogyne incognita. Pest Manag Sci. 2018;74(2):384-391. https://doi.org/10.1002/ps.4717
  7. Scott P, Kennedy B, Van Walbeek W. Desoxypatulinic acid from a patulin-producing strain of Penicillium patulum. Experientia. 1972; 28(10):1252-1252.
  8. Godio R, Fouces R, Gudina E, et al. Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium. Curr Genet. 2004;46(5): 287-294. https://doi.org/10.1007/s00294-004-0533-5
  9. Michielse CB, Hooykaas PJ, van den Hondel CA, et al. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet. 2005;48(1):1-17. https://doi.org/10.1007/s00294-005-0578-0
  10. Ruiz-Diez B. Strategies for the transformation of filamentous fungi. J Appl Microbiol. 2002;92: 189-195. https://doi.org/10.1046/j.1365-2672.2002.01516.x
  11. Frandsen RJ. A guide to binary vectors and strategies for targeted genome modification in fungi using Agrobacterium tumefaciens-mediated transformation. J Microbiol Methods. 2011;87(3): 247-262. https://doi.org/10.1016/j.mimet.2011.09.004
  12. Hansen G, Wright MS. Recent advances in the transformation of plants. Trends Plant Sci. 1999; 4(6):226-231. https://doi.org/10.1016/S1360-1385(99)01412-0
  13. Covert SF, Kapoor P, Lee M-h, et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol Res. 2001; 105(3):259-264. https://doi.org/10.1017/S0953756201003872
  14. Morioka LRI, Furlaneto MC, Bogas AC, et al. Efficient genetic transformation system for the ochratoxigenic fungus Aspergillus carbonarius. Curr Microbiol. 2006;52(6):469-472. https://doi.org/10.1007/s00284-005-0402-6
  15. Liu YG, Mitsukawa N, Oosumi T, et al. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J. 1995;8(3):457-463. https://doi.org/10.1046/j.1365-313X.1995.08030457.x
  16. Ochman H, Gerber AS, Hartl DL. Genetic applications of an inverse polymerase chain reaction. Genetics. 1988;120(3):621-623. https://doi.org/10.1093/genetics/120.3.621
  17. Khang CH, Park SY, Lee YH, et al. A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum. Fungal Genet Biol. 2005;42(6):483-492. https://doi.org/10.1016/j.fgb.2005.03.004
  18. Park S-Y, Jeong M-H, Wang H-Y, et al. Agrobacterium tumefaciens-mediated transformation of the lichen fungus, Umbilicaria muehlenbergii. PLoS One. 2013;8(12):e83896. https://doi.org/10.1371/journal.pone.0083896
  19. Daboussi M, Djeballi A, Gerlinger C, et al. Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans. Curr Genet. 1989;15(6): 453-456. https://doi.org/10.1007/BF00376803
  20. De Groot MJ, Bundock P, Hooykaas PJ, et al. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol. 1998; 16(9):839-842. https://doi.org/10.1038/nbt0998-839
  21. Mullins E, Romaine CP, Chen X, et al. Agrobacterium tumefaciens-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology. 2001;91(2):173-180. https://doi.org/10.1094/PHYTO.2001.91.2.173
  22. Leung H, Lehtinen U, Karjalainen R, et al. Transformation of the rice blast fungus Magnaporthe grisea to hygromycin B resistance. Curr Genet. 1990;17(5):409-411. https://doi.org/10.1007/BF00334519
  23. Punt PJ, van Biezen N, Conesa A, et al. Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol. 2002;20(5): 200-206. https://doi.org/10.1016/S0167-7799(02)01933-9
  24. Winans SC. Two-way chemical signaling in Agrobacterium-plant interactions. Microbiol Rev. 1992;56(1):12-31. https://doi.org/10.1128/mr.56.1.12-31.1992
  25. Bundock P, Hooykaas PJ. Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc Natl Acad Sci USA. 1996; 93(26):15272-15275. https://doi.org/10.1073/pnas.93.26.15272